1. Field
One or more embodiments of the present invention relate to a method of analyzing ubiquitin-proteasome activity and a method of screening a ubiquitin-proteasome inhibitor by using the same.
2. Description of the Related Art
A ubiquitin proteasome system is an important regulatory mechanism in cell growth and division, cell cycle, intracellular signal transduction, and cell apoptosis. Through the regulatory mechanism, a protein acting as a substrate is degraded by a proteasome. In the proteolytic process by the ubiquitin proteasome system, multiple ubiquitin protein chains form a covalent bond with a substrate, and the resulting product is recognized and degraded by a 26S proteasome consisting of a 20S complex and 19S particles. In this process, ubiquitin proteins are bound to a substrate by a ubiquitin-activating enzyme E1, a ubiquitin-conjugating enzyme E2, and a ubiquitin ligase E3, and the resulting ubiquitinated proteins are degraded by a proteasome. Since it is known that the ubiquitin-proteasome system affects the onset of various cancers, neurodegenerative diseases, metabolic disorders, viral diseases, cardiac diseases, and aging-related diseases and the inhibition of proteasome activity suppresses apoptosis and proliferation of cancer cells, there is an increasing interest in the development of a proteasome inhibitor as an anticancer agent.
Recently, as methods of quantitatively observing substrates of the ubiquitin-proteasome system, fluorescent microscopy, flow cytometry, high-throughput screening, pulse chase labelling method, and immunoblotting are used. However, since these methods include many experimental steps, these methods are complicated, time-consuming, and low in quantitative accuracy.
Therefore, there is a need for developing a method of quantitatively analyzing ubiquitin-proteasome activity in a short period of time and a method of screening a proteasome inhibitor.